Project Summary A vaccine against Hepatitis C virus (HCV) is urgently needed. HCV infects over 170 million people worldwide and kills more people in the United States annually than HIV. While direct-acting antiviral (DAA) therapy has revolutionized HCV care, control of the HCV pandemic remains challenging due frequent reinfection in high-risk individuals and a high proportion of asymptomatic carriers who continue to infect others. Approximately 30% of individuals who become infected with HCV spontaneously clear the infection, and we have previously shown that this spontaneous clearance of HCV is associated with early development of broadly neutralizing antibodies (bNAbs) against the virus. BNAbs are also protective against HCV infection in multiple animal models. Unfortunately, to date, vaccines against HCV have not induced adequate titers of protective bNAbs. Our inability to induce potent bNAbs is in part due to our poor understanding of the molecular and structural interactions between bNAbs and HCV envelope proteins (E1 and E2). Our preliminary work indicates that envelope sequence polymorphisms distant from bNAb binding sites have a strong, unexpected influence on neutralization sensitivity. These data and rapidly emerging work in HIV indicate that these crucial bNAb-envelope interactions need to be understood in a three dimensional (structural) context. We hypothesize that molecular and structural analysis of bNAb-E2 interactions will allow us to rationally design stable HCV envelope proteins with optimized bNAb epitopes that are ideal for structural and vaccine studies as well as bNAbs with enhanced neutralizing potency and breadth, better defining the ideal antibodies that should be induced by a vaccine. We have characterized a diverse panel of unique HCV envelope proteins and isolated some of the most broadly neutralizing anti-HCV monoclonal antibodies described to date. In Aim 1, we will functionally and molecularly characterize interactions between this panel of diverse, naturally occurring HCV envelope variants and the panel of bNAbs, which will allow us to identify amino acid determinants of neutralization sensitivity of E2 as well as somatic mutations conferring neutralizing potency and breadth to bNAbs. In Aim 2, we will define biochemical and molecular factors influencing stability and native folding of HCV envelope proteins. We will clone more than 100 distinct natural HCV E2 variants and identify polymorphisms associated with stable in vitro E2 expression. In Aim 3, we will determine structural correlates of broad and potent neutralization of HCV. We will crystallize HCV E2 in complex with bNAbs of varying breadth and potency. We will use the data acquired through these three aims to design stable E2 variants with optimized bNAb epitopes that will be ideal reagents for future structure analyses and vaccine studies. In addition, we will design bNAbs with enhanced neutralizing potency and breadth that will define the ideal antibodies that could be induced by a vaccine and may also be useful therapeutic agents. Through these investigations, we will advance rational design of an HCV vaccine.